KR101363682B1 - Marker protein for type 2 diabetes - Google Patents

Abstract

The present invention provides marker proteins for early detection of type 2 diabetes, antibodies to the marker proteins and diagnostic methods for type 2 diabetes and their use in drug development.

Description

Marker protein for type 2 diabetes {MARKER PROTEIN FOR TYPE 2 DIABETES}

The present invention provides diagnostic marker proteins for the early detection of type 2 diabetes, antibodies to these marker proteins and diagnostic methods for type 2 diabetes and their use in drug development.

Type 2 diabetes (non-insulin dependent diabetes mellitus (NIDDM)) is a disease characterized by high blood glucose in the context of insulin resistance and relative insulin deficiency. There are an estimated 23.6 million people in the United States (7.6% of the total population), of which 17.9 million are diagnosed with diabetes, more than 90% of which are type 2 diabetes. As the prevalence doubled between 1990 and 2005, the Centers for Disease Control and Prevention classified the increase as epidemic levels. Thus, there is a need for diagnostic markers and methods that allow early detection of type 2 diabetes.

The present invention provides diagnostic marker proteins for the early detection of type 2 diabetes, antibodies to these marker proteins and diagnostic methods for type 2 diabetes and their use in drug development.

In one aspect, the invention provides a subject for the diagnosis of type 2 diabetes or for the development of type 2 diabetes comprising measuring the level of Olfactomedin 4 (OLFM4) polypeptide in a tissue sample of the subject. A method of determining the predisposition of, wherein a reduced level of OLFM4 polypeptide in a subject's sample is indicative of predisposition to type 2 diabetes or type 2 diabetes as compared to the level of OLFM4 polypeptide representing a healthy population. It is about how.

In a preferred embodiment, the tissue is blood, preferably plasma.

In a second aspect, the present invention

a) administering the compound to a non-human animal suffering from type 2 diabetes,

b) A method of identifying a compound for treatment of type 2 diabetes, comprising measuring the level of OLFM4 polypeptide in a tissue sample of a non-human animal of step a), wherein the type 2 diabetes is not administered the compound The altered level of OLFM4 polypeptide in the tissue sample of the non-human animal of step a) compared to the OLFM4 polypeptide level in the tissue sample of the non-human animal suffering from is an indicator representing a compound for the treatment of type 2 diabetes. To provide.

In a preferred embodiment, the tissue sample is blood, preferably plasma.

In a further preferred embodiment, the non-human animal is a rodent, preferably a mouse or a rat, more preferably a DIO mouse, ob / ob mouse or ZDF rat.

In a third aspect, the invention relates to the use of an OLFM4 polypeptide for determining the predisposition of an individual to the diagnosis of type 2 diabetes or to the development of type 2 diabetes.

In a preferred embodiment, the OLFM4 polypeptide is a human OLFM4 polypeptide. The amino acid sequence of human OLFM4 is disclosed in SEQ ID NO: 1.

In a fourth aspect, the present invention provides the use of an antibody that specifically binds an OLFM4 polypeptide for the diagnosis of type 2 diabetes or for determining the predisposition of an individual to the onset of type 2 diabetes.

In a preferred embodiment, the antibody binds to a human OLFM4 polypeptide.

In a fifth aspect, the present invention

a) an antibody specific for an OLFM4 polypeptide, preferably an antibody of the invention,

b) a labeled antibody that binds to OLFM4 captured by the antibody of a) or a labeled antibody that binds to the antibody of a), and

c) a kit for diagnosing type 2 diabetes comprising a reagent for performing a diagnostic assay or for determining a predisposition to the development of type 2 diabetes in a subject.

In a preferred embodiment, the antibody specific for the OLFM4 polypeptide binds to a human OLFM4 polypeptide.

The method of the present invention can be used to monitor Type 2 diabetes therapy response in patients by measuring OLFM4 polypeptide levels in tissue samples, preferably blood samples, of patients undergoing diabetes therapy. Patients exhibiting altered levels of OLFM4 polypeptide in tissue samples during the course of therapy relative to OLFM4 polypeptide levels at the beginning of therapy are responding to diabetes therapy.

In yet another aspect, the present invention relates to monoclonal antibodies against human OLFM4 polypeptide.

In a preferred embodiment, the antibody comprises OLFM4 2/3 (DSM ACC3012), OLFM4 1/46 (DSM ACC3011), OLFM4 2/1 (DSM ACC3013), OLFM4 2/14 (DSM ACC3014), OLFM4 2/28 (DSM ACC3015). ) And CDR1 to CDR3 of the V _H domain of an antibody obtainable from a hybridoma cell line selected from the group consisting of OLFM4 1/23 (DSM ACC3010), and OLFM4 2/3 (DSM ACC3012), OLFM4 1/46 (DSM ACC3011) Of an antibody obtainable from a hybridoma cell line selected from the group consisting of OLFM4 2/1 (DSM ACC3013), OLFM4 2/14 (DSM ACC3014), OLFM4 2/28 (DSM ACC3015) and OLFM4 1/23 (DSM ACC3010). An antibody comprising CDR1 to CDR3 of a V _L domain.

In a further embodiment, the antibody is OLFM4 2/3 (DSM ACC3012), OLFM4 1/46 (DSM ACC3011), OLFM4 2/1 (DSM ACC3013), OLFM4 2/14 (DSM ACC3014), OLFM4 2/28 (DSM ACC3015). ) And OLFM4 1/23 (DSM ACC3010) are chimeric antibodies comprising the V _H and V _L domains of an antibody obtainable from a hybridoma cell line selected from the group consisting of.

In a further preferred embodiment, the antibody comprises OLFM4 2/3 (DSM ACC3012), OLFM4 1/46 (DSM ACC3011), OLFM4 2/1 (DSM ACC3013), OLFM4 2/14 (DSM ACC3014), OLFM4 2/28 (DSM ACC3015) and OLFM4 1/23 (DSM ACC3010) are produced by hybridoma cell lines selected from the group consisting of.

Methods of detecting and / or measuring polypeptides in biological samples are well known in the art and include, but are not limited to, Western-blotting, ELISA or RIA, or various proteomic techniques. Monoclonal or polyclonal antibodies that recognize the OLFM4 polypeptide / fragment, or peptide fragment thereof, may be prepared for the purpose of detecting the polypeptide or peptide fragment, for example by immunizing rabbits with purified protein or Or known antibodies that recognize the polypeptide or peptide fragment can be used. For example, antibodies capable of binding to denatured proteins, such as polyclonal antibodies, can be used to detect OLFM4 polypeptides / fragments thereof in western blots. One example of a method for measuring a marker is ELISA. Measurement of the amount of protein in this format is based on an antibody capable of capturing a specific antigen and a second antibody capable of detecting the captured antigen. Methods and assays for making and using the antibodies described above are described in Harlow, E. and Lane, D., Antibodies: A Laboratory Manual (1988), Cold Spring Harbor Laboratory Press.

In a further aspect, the present invention

a) providing a sample of pancreatic tissue from an individual or non-human animal,

b) detecting pancreatic beta-cells in a tissue sample comprising detecting OLFM4 positive cells in a tissue sample of a), wherein the OLFM4 positive cells are beta-cells.

In a preferred embodiment, OLFM4 positive cells are detected by an antibody specific for OLFM4, preferably an antibody of the invention. Methods of detecting beta-cells in tissue samples of human subjects or non-human animals can be used to assess the effect of type 2 diabetes therapy on the physiology / histology of the pancreas. For example, during the development of a compound for the treatment of type 2 diabetes, the method of detecting beta-cells of the present invention may be directed to whether the compound has an effect on the physiology / histology of the pancreas, ie, the compound is resistant to type 2 diabetes. Can be used to assess whether some of the effects of type 2 diabetes can be reversed in the pancreas of the model.

In a further aspect, the present invention

a) an antibody specific for an OLFM4 polypeptide, preferably an antibody of the invention,

b) a labeled antibody that binds to the antibody of a) or a labeled antibody specific for the OLFM4 polypeptide, and

c) Provide a kit for detecting beta-cells in a pancreatic tissue sample comprising reagents for performing immunohistochemical analysis.

Synonyms for polypeptide olactomedin 4 (OLFM4) are hGC-1 and GW112.

As used herein, the term "polypeptide" refers to a polymer of amino acids that is not of a particular length. Thus, peptides, oligopeptides and protein fragments are included within the definition of polypeptide.

The term “compound” is used herein in the context of “test compound” or “drug candidate compound” as described in connection with the assay of the present invention. Therefore, these compounds include organic or inorganic compounds derived synthetically or from natural sources. Compounds include inorganic or organic compounds such as polynucleotides, lipids or hormonal analogs characterized by relatively low molecular weight. Other biopolymer organic test compounds include peptides comprising about 2 to about 40 amino acids and large polypeptides comprising about 40 to about 500 amino acids, such as antibodies or antibody conjugates.

The term “antibody” includes various forms of antibody structures, including but not limited to whole antibodies and antibody fragments. Antibodies according to the invention are preferably humanized antibodies, chimeric antibodies or additionally genetically engineered antibodies which retain the characteristic properties according to the invention.

An “antibody fragment” comprises a portion of an antibody of full length, preferably its variable domain, or at least its antigen binding site. Examples of antibody fragments include diabodies, single-chain antibody molecules and multispecific antibodies formed from antibody fragments. scFv antibodies are described, for example, in Houston, JS, Methods in Enzymol. 203 (1991) 46-96. In addition, antibody fragments of, or V _H domain and a function having a characteristic of the V _L domain that binds the single-chain polypeptide, which may be combined with, or V _L domain having the characteristics of a V _H domain, or the ANG-2 Single chain polypeptides that can be bound to antigen binding sites to provide properties.

The term "monoclonal antibody" or "monoclonal antibody composition" as used herein refers to the preparation of an antibody molecule of a single amino acid composition.

The term “chimeric antibody” refers to an antibody comprising variable regions from one source or species, usually binding sites and at least a portion of constant regions derived from different sources or species, usually produced by recombinant DNA technology. Chimeric antibodies comprising murine variable regions and human constant regions are preferred. Another preferred form of "chimeric antibody" encompassed by the present invention is that the constant region of the original antibody is modified or changed, in particular exhibiting properties according to the invention for Clq binding and / or Fc receptor (FcR) binding. Such chimeric antibodies are also referred to as "class-switched antibodies". Chimeric antibodies are the product of expressed immunoglobulin genes comprising a DNA portion encoding an immunoglobulin variable region and a DNA portion encoding an immunoglobulin constant region. Methods for making chimeric antibodies employ conventional recombinant DNA, and gene transformation techniques are well known in the art. For example, Morrison, S.L., et al., Proc. Natl. Acad. Sci. USA 81 (1984) 6851-6855; See US Pat. Nos. 5,202,238 and 5,204,244.

The term "human antibody" as used herein is intended to include antibodies having variable and constant regions derived from human germ line immunoglobulin sequences. Human antibodies are well known in the art (van Dijk, M.A., and van de Winkel, J.G., Curr. Opin. Chem. Biol. 5 (2001) 368-374). Human antibodies can also be produced in transgenic animals (eg mice), which can produce the entire repertoire or selection of human antibodies upon immunization, in the absence of endogenous immunoglobulin production.

Migration of human germline immunoglobulin gene sequences into germline mutant mice results in the production of human antibodies following antigen challenge (eg, Jakobovits, A., et al., Proc. Natl. Acad.Sci. USA 90 (1993) 2551-2555; Jakobovits, A., et al., Nature 362 (1993) 255-258; Bruggemann, M., et al., Year Immunol. 7 (1993) 33-40 Reference). Human antibodies can also be produced in phage display libraries (Hoogenboom, HR, and Winter, G., J. Mol. Biol. 227 (1992) 381-388; Marks, JD, et al. J. Mol. Biol. 222 (1991) 581-597). See also Cole et al. And the technique of Boerner et al. Can also be used to prepare human monoclonal antibodies (Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985); and Boerner, P., et. al, J. Immunol. 147 (1991) 86-95). As already mentioned for the chimeric and humanized antibodies according to the invention, the term "human antibody" as used herein is altered at constant regions, especially for Clq binding and / or FcR binding, for example "class-conversion". "I.e., antibodies that exhibit properties according to the invention by changes or mutations in the Fc portion (e.g., from IgGl to IgG4 and / or IgGl / IgG4 mutations).

The term “epitope” includes any polypeptide determinant that enables specific binding to an antibody. In certain embodiments, epitope determinants include chemically active surface groups of molecules such as amino acids, sugar side chains, phosphoryls or sulfonyls, and in certain embodiments, specific three-dimensional structural properties and / or specific charge properties Can have An epitope is the site of an antigen that is bound by an antibody.

As used herein, “variable domain” (variable domain of light chain (V _L ), variable domain of heavy chain (V _H )) refers to a pair of respective light and heavy chain domains that are directly involved in binding of the antibody to the antigen. The variable light and heavy chain domains have the same general structure and each domain is four conserved in sequence and linked by three “hypervariable regions” (or complementary determining regions (CDRs)). Frame work (FR) sites. The skeletal region employs a beta-sheet structure, and the CDRs may form a loop connecting the beta-sheet structure. The CDRs of each chain are maintained in their three-dimensional structure by the backbone sites and together with the CDRs from the other chain form an antigen binding site. The heavy and light chain CDR3 sites of the antibody play a particularly important role in the binding specificity / binding affinity of the antibody according to the invention, thus providing additional aspects of the invention.

As used herein, the term “antigen-binding portion of an antibody” refers to the amino acid residue of an antibody that is responsible for antigen-binding. The antigen-binding portion of an antibody includes amino acid residues from "complementarity determining sites" or "CDRs." A "skeleton" or "FR" site is a variable domain site that is not a hypervariable site as defined herein. Thus, the light and heavy chain variable domains of an antibody comprise the FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4 domains from the N-terminus to the C-terminus direction. In particular, CDR3 of the heavy chain is the site most contributing to antigen binding and defines antibody properties. CDR and FR sites are from the standard definition and / or "overvariable loops" of Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991). It depends on the residue.

Monoclonal antibodies can be prepared using hybridoma methods such as those described in Kohler and Milstein, Nature 256: 495 (1975). In the hybridoma method, mice, hamsters, or other suitable host animals are typically immunized with an immunizing agent to induce lymphocytes that can produce or produce antibodies that will specifically bind to the immunizing agent.

Any of a number of well-known protocols used to fuse lymphocytes and immortalized cell lines can be applied for the purpose of producing monoclonal antibodies of the invention (eg, G. Galfre et al. (1977) Nature. 266: 55052; Gefter et al. Somatic Cell Genet., Cited above; Lerner, Yale J. Biol. Med., Cited above; Kenneth, Monoclonal Antibodies, cited above). Those skilled in the art will also recognize that there are many variations of this method that are useful. Immortal cell lines (eg, myeloma cell lines) are typically derived from mammalian species with the same lymphocytes. For example, murine hybridomas can be prepared by fusing lymphocytes from mice immunized with the immunological agents of the invention with immortal mouse cell lines. Preferred immortalized cell lines are mouse myeloma cell lines that are sensitive to culture medium (“HAT” medium) comprising hypoxanthine, aminopterin and thymidine. Any number of myeloma cell lines, such as P3-NSl / l-Ag4-l, P3-x63-Ag8.653 or Sp2 / 0-Agl4 myeloma cell lines, can be used as fusion partners according to standard techniques. Such myeloma cell lines can be obtained from ATCC. Typically, HAT-sensitive mouse myeloma cells are fused to mouse splenocytes using polyethylene glycol (“PEG”). Hybridoma cells generated by fusion are then selected using HAT medium to kill unfused or non-productive fused myeloma cells (unfused splenocytes die after a few days because they were not transformed). Hybridoma cells producing monoclonal antibodies of the invention are detected by screening hybridoma culture supernatants, for example, for binding antibodies using standard ELISA assays.

Antibodies can be prepared using, for example, recombinant methods and compositions described in US Pat. No. 4,816,567. In one embodiment, an isolated nucleic acid encoding an anti-OLFM4 antibody described herein is provided. Such nucleic acids may encode an amino acid sequence comprising the V _L of an antibody and / or an amino acid sequence comprising the V _H of an antibody (eg, the light and / or heavy chains of the antibody). In other embodiments, one or more vectors (eg, expression vectors) comprising such nucleic acids are provided. In other embodiments, host cells comprising such nucleic acids are provided. In one such embodiment, the host cell comprises (1) a vector comprising an amino acid sequence comprising the V _L of the antibody and a nucleic acid encoding an amino acid sequence comprising the V _H of the antibody, or (2) the V _L of the antibody A first vector comprising a nucleic acid encoding an amino acid sequence and a second vector comprising a nucleic acid encoding an amino acid sequence comprising a V _H of an antibody (eg, is transformed). In one embodiment, the host cell is a eukaryotic cell, eg, a Chinese hamster ovary (CHO) cell or a lymphatic system cell (eg, YO, NSO, Sp20 cell). In one embodiment, culturing the host cell comprising a nucleic acid encoding the antibody as provided above under conditions appropriate for expression of the antibody and optionally recovering the antibody from the host cell (or host cell culture medium). A method of preparing an anti-TMEM27 antibody is provided.

For recombinant production of the antibodies of the invention, for example, nucleic acids encoding the antibodies as described above are isolated and inserted into one or more vectors for further cloning and / or expression in a host cell. Such nucleic acids can be readily isolated and sequenced using conventional methods (eg, by using oligonucleotide probes that can specifically bind to the genes encoding the heavy and light chains of the antibody). .

Suitable host cells for cloning or expression of antibody-encoding vectors include prokaryotic or eukaryotic cells described herein. For example, antibodies can be prepared in bacteria, especially when glycosylation and Fc effector function are not needed. For expression of antibody fragments and polypeptides in bacteria, see, eg, US Pat. Nos. 5,648,237, 5,789,199, and 5,840,523 (also Charlton, Methods describing the expression of antibody fragments in E. coli). in Molecular Biology, Vol. 248 (BKC Lo, ed., Humana Press, Totowa, NJ, 2003), pp. 245-254). After expression, the antibody can be isolated from bacterial cell paste in soluble fraction and further purified.

Methods for cloning antibody genes from hybridoma cells that produce monoclonal antibodies are known to those skilled in the art. For example, genetic information on the variable heavy and light chain domains (V _H and V _L ) can be amplified from hybridoma cells using polymerase chain reaction (PCR) with immunoglobulin-specific primers. Mol Med. 2004; 94: 447-58). Nucleic acids encoding variable heavy and light chain domains (V _H and V _L ) can then be cloned into a vector suitable for expression in a host cell.

The marker protein of the present invention and the antibody to the marker protein can be used for the diagnosis of diabetes or a method for determining the predisposition of an individual for the onset of diabetes and the development of a compound for treating diabetes.

1A shows the results of detecting OLFM4 polypeptides in 10 human plasma samples by ELISA using antibody pairs OLFM4-1/23 and OLFM4-2/3.
1B shows the results of detecting OLFM4 polypeptides in 10 human plasma samples by ELISA using antibody pairs OLFM4-2 / 1 and OLFM4-2 / 28.
1C shows the results of detecting OLFM4 polypeptides in 10 human plasma samples by ELISA using antibody pairs OLFM4-2 / 28 and OLFM4-2 / 14.
2A shows the results of immunoprecipitation (IP) in 10 human plasma samples using monoclonal antibody OLFM4-2 / 1.
2B shows the results of immunoprecipitation (IP) in 10 human plasma samples using monoclonal antibody OLFM4-2 / 3.
2C shows the results of immunoprecipitation (IP) in 10 human plasma samples using monoclonal antibody OLFM4-2 / 28.
2D shows the results of immunoprecipitation (IP) in 10 human plasma samples using monoclonal antibody OLFM4-2 / 14.
3A shows healthy controls, fasting glucose impairment (IFG), impaired glucose tolerance (IGT), fasting glucose impairment + impaired glucose tolerance (IFG + IGT) by ELISA using antibody pairs OLFM4 2/1 and OLFM4 2/28 , OLFM4 polypeptide is detected in plasma samples of human subjects selected from the group of type 1 diabetic patients (T1D) and type 2 diabetic patients (T2D).
3B shows healthy controls, fasting glucose disorders (IFG), impaired glucose tolerance (IGT), fasting glucose disorders + impaired glucose tolerance (IFG + IGT) by ELISA using antibody pairs OLFM4 2/28 and OLFM4 2/14 , OLFM4 polypeptide is detected in plasma samples of human subjects selected from the group of type 1 diabetic patients (T1D) and type 2 diabetic patients (T2D).
4A shows the result of staining a human tissue array by immunohistochemistry (IHC) using the monoclonal antibody hOLFM4 1/46.
4B and 4C show human pancreatic islets stained with monoclonal antibody hOLFM4 1/46 (OLFM4: green, glucagon: red, DAPI: blue).

Example

The Monoclonal  Anti human OLFM4  Antibody

The following five mouse hybridoma cell lines that produce monoclonal antibodies against human OLFM4, in the name of F. Hoffmann-La Roche Ltd., on October 7, 2009, in Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ). It was deposited and received the access numbers listed below:

OLMF4-1 / 23 = DSM ACC3010

OLMF4-1 / 46 = DSM ACC3011

OLMF4-2 / 3 = DSM ACC3012

OLMF4-2 / 1 = DSM ACC3013

OLMF4-2 / 14 = DSM ACC3014

OLMF4-2 / 28 = DSM ACC3015

human OLFM4 Mouse for Monoclonal  Generation of Antibodies (Mouse OLFM4 mAbs )

The amino acid sequence of the recombinant human OLFM4 fusion polypeptide used to produce the monoclonal antibody is given below:

GSGGSVSQLFSNFTGSVDDRGTCQCSVSLPDTTFPVDRVERLEFTAHVLSQKFEKE

LSKVREYVQLISVYEKKLLNLTVRIDIMEKDTISYTELDFELIKVEVKEMEKLVIQL

KESFGGSSEIVDQLEVEIRNMTLLVEKLETLDKNNVLAIRREIVALKTKLKECEASK

DQNTPVVHPPPTPGSCGHGGVVNISKPSVVQLNWRGFSYLYGAWGRDYSPQHPN

KGLYVAPLNTDGRLLEYYRLYNTLDDLLLYINARELRTYGQGSGTAVYNNNMYV

NMYNTGNIARVNLTTNTIAVTQTLPNAAYNNRFSYAVAWQDIDFAVDENGLWVI

YSTEASTGNMVISKLNDTTLQVLNTWYTKQYKPSASNAFMVCGVLYATRTMNTR

TEEIFYYDTNTGKEGKLDIVMHKMQEKVQSINYNPFDQLYVYNDGYLL

NYDLSVLQKPQHHHHHH (SEQ ID NO: 2)

Mice were immunized with 5 μg / injection of recombinant OLFM4 coupled to His tag, produced in insect cells. ImmunEasy adjuvant (ALHY-DROGEL 2% + CPG-ODN) was administered intraperitoneally on days 0, 13 and 28 of immunization. Evaluation of the immune response of the animals was performed by ELISA for recombinant OLFM4 with blood collected on day 41. Superboost (intravenous administration of 5 μg of recombinant OLFM4 in PBS) was performed on day 56 in two selected animals and PAI-cells and spleen cells were fused 2 days later. Hybridoma screening and cloning assessments were performed by ELISA for recombinant OLFM4.

ELISA  Specificity check

Three pairs of mAbs of the invention were used for ELISA.

[Table 1]

Of 10 control human plasma ELISA  result

The analysis results are shown in FIGS. 1A-1C:

Hu 1-Hu 10: human serum control (blood donor human plasma)

Positive Control (OLFM4): INS-1 hOLFM4 WT F11

Negative Control (med): Medium

The use of three different ELISA assays to test 10 human plasma samples gave very similar results. This demonstrates the specificity of the assay. The samples were further used to qualitatively verify the ELISA results by immunoprecipitation (IP).

IP On by ELISA Qualitative confirmation (immunoprecipitation method ( IP ) - mAb  Final Selection of Pairs: Blood Donor and Human Plasma INS -One hOLFM4 WT  F11 / badge)

Immunoprecipitation (IP) was performed on human plasma samples used in previous ELISAs to assess whether similar results were obtained by different techniques (see FIGS. 2A-2D). There was a perfect match between the ELISA and IP results. This is very important as it is a qualitative confirmation of the results using two different techniques.

The following samples and antibodies were used for IP experiments:

Sample:

[Table 2]

MWM: molecular weight marker; The control was identical to the ELISA assay.

Antibodies:

Fig. 2a: OLFM4-2/1

Fig. 2b: OLFM4-2/3

Fig. 2c: OLFM4-2/28

Fig. 2d: OLFM4-2/14

The results of the IP analysis are shown in Figures 2A-2D:

Samples positive in ELISA (# 1-5 and # 7-10) were also positive in IP. Samples that were negative in ELISA (# 6) were also negative in IP. INS-1 OLFM4 supernatants and media were used as positive and negative controls, respectively. This is a qualitative confirmation of the ELISA results by IP.

Human cross-section population ( cross sectional cohort From ELISA  Results: Significantly decreased in pre-diabetic and diabetic patients. OLFM4  (Bratislava group). 3a + 3b

Human plasma population

 Object Screening

About 200 individuals with metabolic risk of T2D enrolled in an outpatient clinic meeting the following inclusion criteria received an oral glucose tolerance test (75 g):

Gender: male

Age: 40-55

BMI: 25-32 kg / m2

HbA1C <7.0%.

Exclusion criteria included prior knowledge of changes in glucose metabolism, the use of drugs known to alter insulin secretion or activity, and the presence of liver or endocrine diseases. Prior to collection of blood samples, height and weight were measured using standard protocols. Body mass index (BMI) was calculated by dividing weight in kilograms by height in meters squared. After 10-12 hours overnight fasting and 2 hours after glucose administration, whole blood samples (20 ml) were collected from EDV test tubes into the EDTA test tube. In order to obtain an adequate fasting state, participants were provided with accurate guidance on the type of food and their last ingestion time. Plasma separated by centrifugation was stored at −80 ° C. in 1 ml aliquots (10 ×) prior to analysis. A informed consent was signed and patients meeting the criteria were enrolled in this study. The Ethics Committee of the Experimental Endocrinology Association of Slovak Academy of Sciences approved the protocol for this experiment.

Diagnosis

ADA Guidelines 2005 ( Dibetes Care . Subjects were classified into four different groups according to 2005 Jan; 28 Suppl 1: S37-42):

Healthy Control : Fasting Plasma Glucose (FPG) <5.6 mmol / l and Normal Glucose Tolerance (NGT) <7.8 mmol / l.

Fasting Glucose Disorder ( IFG ) : IFG was defined as an FPG value equal to or greater than 5.6 mmol / l and less than 6.9 mmol / l and normal glucose tolerance (NGT) less than 7.8 mmol / l 2 hours after administration.

Glucose Tolerance Disorder ( IGT ) : IGT was defined as the glucose concentration 2 hours after administration equal to or greater than 7.8 mmol / l and less than 11.1 mmol / l.

Fasting glucose and glucose tolerance disorders ( IFG + IGT ) : IGT + IFG is equal to or greater than 5.6 mmol / l and less than 6.9 mmol / l and FPG value greater than or equal to 7.8 mmol / l after 2 hours and greater than 11.1 mmol / l It is defined as a small NGT value.

In addition, groups of 8 type 1 diabetics and 11 type 2 diabetic patients also have an outpatient clinic with patients with dyslipidemia treated with dyslipidemia drugs (eg, statins or fibrates). Selected from the list.

Diabetes of individuals Summary table

[Table 3]

Average of individuals with type 1 diabetes (T1-DM), type 2 diabetes (T2-DM), glucose tolerance disorder (IGT), fasting glucose disorder (IFG), glucose tolerance disorder (IGT), and IFG + IGT Humanistic side and test result characteristics

[Table 4]

The following OLFM4 monoclonal antibody pairs were used for ELISA analysis.

Figure 3a: Antibody: 2/1-2/28

Figure 3b: Antibodies: 2/28-2/14

ELISA results in the cross-particulate group showed significantly lower OLFM4 levels in pre-diabetic patients (IFG + IGT, IFG, and IGT) compared to healthy control patients (FIGS. 3A and 3B). In addition, OLFM4 levels in T2DM patients were low. Interestingly, OLFM4 levels in T1DM patients were high but not significant (ANOVA with Dunnett's correction). Both T2DM and T1DM patient groups were being treated.

Given that OLFM4 is significantly reduced in pre-diabetic patients who are not being treated, we believe that OLFM4 can be used as a marker for the onset of early T2D disease.

For pancreatic beta-cells As a marker OLFM4

Immunohistochemical (IHC) staining of human tissue arrays using the monoclonal antibody hOLFM4 1/46. Very strong and specific signals were detected in the beta-cells of the human islet (human pancreatic part), while the results did not show specific staining in any of the tissues tested. The pancreatic part of the tissue microarray was negative because there was no islet structure but only exocrine gland tissue at the pancreatic point of the tissue microarray.

4A: Human tissue array stained with monoclonal antibody hOLFM4 1/46

4B and 4C: Human pancreatic islets stained with monoclonal antibody hOLFM4 1/46 (OLFM4: green, glucagon: red, DAPI: blue).

Materials and methods

ELISA  protocol

coating:

Coating-mAb: 5 μg / ml in PBS, 100 μl / well

-> Overnight in a humid box at 4 ℃

-> 2x Wash PBS-Tween

blocking:

B-buffer

200 μl / well

-> 1 hour at 37 ℃

-> 2x Wash PBS-Tween

Sample and Detection mAb :

Biotinylated detection in 1 μg / ml B-buffer-mAb: 25 μl / well

Sample (human plasma): diluted in B-buffer, starting with undiluted plasma, 30 μl / well from 1: 2 to 1: 128 (8 concentrations)

First 25 μl of Detection-mAb is added to the plate followed by 30 μl of sample.

-> Overnight in a humid box on a shaker at 4 ° C

-> 4x Wash PBS-Tween

concrete( conjugate ):

PIERCE streptavidin-HRPO in 1 μg / ml B-buffer (No 21126)

50 μl / well

-> 1 hour at room temperature

-> 4x Wash PBS-Tween

temperament( 기판 ):

3,3 ', 5,5'-tetramethylbenzidine (TMB), 100 μl / well

Reaction with 0.5MH ₂ SO ₄ after 5 min, 100 μl / well

Measured at 450nm

Cell culture

Doxycline-inducible rat insulin species INS-1 hOLFM4 WT and INS-1 hOLFM4-His stable cell lines (wild type (hOLFM4 WT) and His tagged (hOLFM4-His) human OLFM4 form expression, respectively) were previously described. Incubated as described (Wang et al. 2001). Two INS-1 cell lines were loaded with 10 mM Hepes (pH 7.4), 1 mM sodium pyruvate, 50 μM 2-mercaptoethanol, 10% heat-inactivated fetal bovine serum (FBS), penicillin, and streptoto Growing in RPMI 1640 + GlutaMAX-1 medium (Invitrogen, Carlsbad, Calif.) Containing mycin. 50 μg / ml G418 sulphate (Promega, Madison, Wis.) And 50 μg / ml zeocin (Invitrogen) were added for growth selection. Overexpression of hOLFM4 WT and hOLFM4-His was induced by 500 ng / ml of Doxycycle (Dox) (Sigma) for 96 hours. Cells were grown in a humidified incubator at 37 ° C. and 5% CO ₂ .

Immunoprecipitation ( IP ) And immunity Blasting ( Western Blat , WB )

Cells of 60-90% density were incubated in 10 cm Petri dishes for 96 hours in the presence or absence of 500 ng / ml doxycycle. Supernatants (cell culture medium) were harvested under sterile conditions, centrifuged at 2000 rpm for 10 minutes and stored at 4 ° C. Cells were washed twice in 1 × PBS and lysed with 1 mL lysis buffer. After 5 minutes, cells were collected in 1.5 mL Eppendorf tubes and centrifuged for 5 minutes at full speed. Supernatants (whole cell extracts) were collected, divided into portions, frozen in liquid nitrogen and stored at -80 ° C. For IP, 3 mL of supernatant (cell culture medium) was mixed with 1 μg of each mAb and incubated for 48 hours at 4 ° C., an orbital shaker. 25 μl of Protein A Sepharose CL-4B diluted 50% in 1 × PBS-Tween (0.05%) was added to each reaction and incubated for 1 hour at room temperature on an orbital shaker. The tube was spin-down and the precipitate was washed twice with 1 × PBS-Tween (0.05%) and once with 1 × PBS. 35 μl of 1 × LDS-SB / 10% beta-ME was added to each precipitate and the samples were vigorously vortexed and spun down before loading onto the SDS-PAGE gel. Immunoblotting using improved chemiluminescence (Pierce, Rockford, IL, USA) for detection was performed as previously described (Wang H, J Biol Chem 2001).

Immunohistochemistry IHC )

Formalin-fixed, paraffin-embedded (FFPE) fragments were used as assemble slides. Samples were dehydrated and the slides were sequentially immersed in xylol (x2), 100% ethanol, 95% ethanol, 80% ethanol, 70% ethanol, and 1X PBS (3 minutes each). Antigen recovery was performed by immersing the slide in 1 × citrate buffer and boiling it for 3 minutes in the microwave (850 watts). Slides were washed twice with water and cells were treated with 100 μl of 0.2% Triton in 1 × PBS for 10 minutes at room temperature. After washing three times with IX PBS, blocking was performed with 2% BSA in IX PBS for 30 minutes to 1 hour at room temperature. Three additional washes with IX PBS were performed and primary Ab incubation (1-2 hours at 37 ° C or overnight at 4 ° C). After three additional washes with IX PBS, secondary Ab incubation was performed for 1 hour in the dark at room temperature. After three additional washes, DAPI staining (5-10 minutes at room temperature in the dark). The cover slip was put on after three final washes.

FDA standard human tissue microarrays (T8234700, Biochain) were treated with mouse anti-OLFM4 monoclonal antibodies, followed by staining with Alexa 488 conjugated donkey anti-mouse and Alexa 555 donkey anti-talkie secondary antibody (Invitrogene).

Human pancreatic fragments obtained from Asterand were co-stained with mouse anti-OLFM4 monoclonal antibodies and rabbit anti-glucagon polyclonal antibodies, and Alexa 488 conjugated donkey anti-mouse and Alexa 555 donkey anti-rabbit secondary Staining with antibody (Invitrogene).

While preferred embodiments of the invention have been described and shown, it is to be understood that the invention is not limited thereto but may be embodied and practiced in various ways within the scope of the following claims.

DSMZ DSMACC03010 20091007 DSMZ DSMACC03011 20091007 DSMZ DSMACC03012 20091007 DSMZ DSMACC03013 20091007 DSMZ DSMACC03014 20091007 DSMZ DSMACC03015 20091007

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Claims (24)

A method of measuring the level of olpactodine 4 (OLFM4) polypeptide in a tissue sample of an individual to collect information for diagnosing the type 2 diabetes or for determining the predisposition of the individual to the onset of type 2 diabetes. Reduced level of OLFM4 polypeptide in a subject's sample compared to the level of OLFM4 polypeptide representative of is an indicator of prevalence of type 2 diabetes or type 2 diabetes and the tissue sample is blood. A method of identifying a compound for the treatment of type 2 diabetes,
a) administering the compound to a non-human animal suffering from type 2 diabetes, and
b) measuring the level of OLFM4 polypeptide in the tissue sample of the non-human animal of step a), wherein the OLFM4 poly in the tissue sample of the non-human animal suffering from type 2 diabetes without the compound The altered level of OLFM4 polypeptide in the tissue sample of the non-human animal of step a) compared to the peptide level is an indicator of a compound for the treatment of type 2 diabetes, and the tissue sample is blood. The method of claim 2, wherein the non-human animal is rodent. The method of claim 3, wherein the rodent is a ZDF rat or ob / ob mouse. a) an antibody specific for the OLFM4 polypeptide,
b) a labeled antibody that binds to the antibody of a) or a labeled antibody that binds to the captured OLFM4 polypeptide of a), and
c) reagents for performing diagnostic analysis
Kit for performing the method of claim 1 comprising a. The kit of claim 5, wherein the antibody specific for the OLFM4 polypeptide binds a human OLFM4 polypeptide. delete The method of claim 1, wherein a monoclonal antibody against human OLFM4 polypeptide is used to determine the level of OLFM4 polypeptide in a subject's tissue sample, wherein the antibody is OLFM4 2/3 (DSM ACC3012), OLFM4 1/46 ( From hybridoma cell lines selected from the group consisting of DSM ACC3011), OLFM4 2/1 (DSM ACC3013), OLFM4 2/14 (DSM ACC3014), OLFM4 2/28 (DSM ACC3015) and OLFM4 1/23 (DSM ACC3010). CDR1 to CDR3 of the V _H domain of the obtainable antibody, and OLFM4 2/3 (DSM ACC3012), OLFM4 1/46 (DSM ACC3011), OLFM4 2/1 (DSM ACC3013), OLFM4 2/14 (DSM ACC3014), OLFM4 2/28 (DSM ACC3015) and OLFM4 1/23 (DSM ACC3010) comprising CDR1 to CDR3 of the V _L domain of an antibody obtainable from a hybridoma cell line selected from the group consisting of: The method of claim 1, wherein a monoclonal antibody against human OLFM4 polypeptide is used to determine the level of OLFM4 polypeptide in a subject's tissue sample, wherein the antibody is OLFM4 2/3 (DSM ACC3012), OLFM4 1/46 ( Obtained from a hybridoma cell line selected from the group consisting of DSM ACC3011), OLFM4 2/1 (DSM ACC3013), OLFM4 2/14 (DSM ACC3014), OLFM4 2/28 (DSM ACC3015) and OLFM4 1/23 (DSM ACC3010). A method comprising the V _H domain and the V _L domain of a possible antibody. The method of claim 1, wherein a monoclonal antibody against human OLFM4 polypeptide is used to determine the level of OLFM4 polypeptide in a subject's tissue sample, wherein the antibody is OLFM4 2/3 (DSM ACC3012), OLFM4 1/46 ( Hybridoma cell lines selected from the group consisting of DSM ACC3011), OLFM4 2/1 (DSM ACC3013), OLFM4 2/14 (DSM ACC3014), OLFM4 2/28 (DSM ACC3015) and OLFM4 1/23 (DSM ACC3010). Produced by the method. delete A method of detecting pancreatic beta-cells in a tissue sample of an individual in order to provide the necessary information for diagnosis of type 2 diabetes or for determining the predisposition of the individual to the onset of type 2 diabetes,
a) providing a pancreatic tissue sample of an individual or non-human animal, and b) detecting OLFM4 positive cells in the tissue sample of a), wherein said OLFM4 positive cells are beta-cells. The method of claim 12, wherein the OLFM4 positive cells are detected by an antibody specific for OLFM4. A kit for performing the method of claim 12 comprising a) an antibody specific for an OLFM4 polypeptide, b) a labeled antibody that binds to the antibody of a), and c) a reagent for performing an immunohistochemical analysis. delete delete delete delete delete delete delete delete delete delete

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